Molecular phylogenetic analysis of actin genic regions fromAchlya bisexualis (Oomycota) andCostaria costata (Chromophyta)

Summary Actin genic regions were isolated and characterized from the heterokont-flagellated protists,Achlya bisexualis (Oomycota) andCostaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in theC. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship ofA. bisexualis andC. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogenetic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.     

Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor.

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.     

The source of calcium for CCK-induced contraction of the guinea-pig gall bladder.

The sources of calcium for cholecystokinin octapeptide (CCK-OP)-induced gallbladder smooth muscle contraction are considered both extracellular and intracellular, but the relative need for intracellular calcium especially at low, physiological concentrations is not clear. To better define the calcium sources responsible for guinea-pig gallbladder contractions in vitro, we inhibited calcium influx using the calcium channel blocker, methoxyverapamil, and a calcium-free Krebs' solution. Availability and release of intracellular calcium stores were depleted by strontium substitution and ryanodine. CCK-OP was compared to bethanechol and potassium chloride (KCl). Preventing calcium influx with 10(-5) M methoxyverapamil depressed the responses to CCK-OP, bethanechol and KCl. Methoxyverapamil, however, had little effect on the time-dependent generation of tension to CCK-OP, but significantly reduced the response to bethanechol and KCl, each at ED50. The duration of the contractile response in the calcium-free Krebs' solution to CCK-OP was longer than that for bethanechol. Strontium (2.5 mM) significantly attenuated the response to CCK-OP and bethanechol, but not to KCl. Ryanodine significantly reduced contractions induced by CCK-OP but not for bethanechol, both at low dose ED25. These results indicate that contraction of the guinea-pig gallbladder induced by CCK-OP, bethanechol and KCl requires extracellular calcium influx. Further, the initiation and maintenance of contraction by CCK-OP and bethanechol necessitates calcium mobilisation from intracellular stores. CCK-OP may have a greater penchant for these calcium stores, particularly at physiological doses.     

N-terminal sequence similarities between components of the multicatalytic proteinase complex

The multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase which is composed of many different types of subunit. As part of a study of the possible relationships between subunits, polypeptides derived from the multicatalytic proteinase from rat liver have been subjected to N-terminal amino acid sequence analysis. Although several of the subunits are blocked at their N-termini, sequences have been obtained for 7 of the polypeptides. Each of the 7 sequences is unique but they show considerable sequence similarity, suggesting that the proteins are encoded by members of the same gene family.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.     

Translocation C:D involving chromosomes 11 and 14

Summary A translocation of material from chromosome 11 to chromosome 14 was identified in a 7-month-old male with microcephaly and developmental delay. The break-points appear to be on the long arm of chromosome 11, close to the centromere, and on the short arm of the 14.     

Self-monitoring of blood glucose in diabetic pregnancy.

Admission to hospital is usually recommended to achieve the best possible diabetic control during pregnancy. We have used blood glucose monitoring at home to find out if patients can achieve equally good control outside hospital. Twenty-five consecutive diabetic patients were studied, of whom 20 had taken insulin before pregnancy. Six of their 14 previous pregnancies had ended in perinatal death. The 25 women performed 4247 blood glucose measurements during their pregnancies. Overall the mean blood glucose concentration was 7.1 mmol/l (128 mg/100 ml); before meals the mean was 6.5 mmol/l (117 mg/100 ml). Mean concentrations were lower in the third trimester, but at no stage was control in hospital significantly better than at home. The mean hospital stay before delivery was 22 days, and all patients had live babies. Monitoring blood glucose concentrations at home produces greater understanding and motivation among patients, improves control early in pregnancy, and shortens time spent in hospital.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.